The invention relates to the field of molecular biology and microbiology. More specifically, the invention relates to genes involved in the conversion of hexose sugars into 3-carbon metabolites in methanotrophic bacteria.
Methanotrophic bacteria are defined by their ability to use methane as their sole source of carbon and energy. Although methanol is an obligate intermediate in the oxidation of methane, the ability to grow on methanol alone is highly variable among the obligate methanotrophs (Green, Peter. Taxonomy of Methylotrophic Bacteria. In: Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell and Howard Dalton eds. 1992 Pleanum Press NY. pp. 23-84)). The conversion of C1 compounds to complex molecules with Cxe2x80x94C bonds is difficult and expensive by traditional chemical synthetic routes. Traditionally, methane is first converted to synthesis gas which is then used to produce other small molecular weight industrial precursors. The basic problem is activation of the methane molecule, a process which is thermodynamically very difficult to achieve by chemical means. Methanotrophs have proved useful mediators of this problem.
Methane monooxygenase is the enzyme required for the primary step in methane activation and the product of this reaction is methanol (Murrell et al., Arch. Microbiol. (2000), 173(5-6), 325-332). This remarkable reaction occurs at ambient temperatures and pressures, whereas chemical transformation of methane to methanol requires temperatures of hundreds of degrees and high pressures (Grigoryan, E. A., Kinet. Catal. (1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No. 5,750,821). It is this ability to transform methane under ambient conditions, along with the abundance of methane, that makes the biotransformation of methane a potentially unique and valuable process.
The commercial applications of biotransformation of methane have historically fallen broadly into three categories: 1) Production of single cell protein, (Villadsen, John, Recent Trends Chem. React. Eng., [Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East, New Delhi, India; Naguib, M., Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ. Arab Pet. Exporting Countries, Kuwait, Kuwait); 2) epoxidation of alkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3) biodegradation of chlorinated pollutants (Tsien et al., Gas, Oil, Coal, Environ. Biotechnol. 2, [Pap. Int. IGT Symp. Gas, Oil, Coal, Environ. Biotechnol.], 2nd (1990), 83-104, Editor(s): Akin, Cavit; Smith, Jared. Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9633821; Merkley et al., Biorem. Recalcitrant Org., [Pap. Int. In Situ On-Site Bioreclam. Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson, Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio; Meyer et al., Microb. Releases (1993), 2(1), 11-22). Only epoxidation of alkenes has experienced little commercial success due to low product yields, toxicity of products and the large amount of cell mass required to generate product.
Methanotrophic cells can further build the oxidation products of methane (i.e. formaldehyde) into more complex molecules such as protein, carbohydrate and lipids. For example, under certain conditions methanotrophs are known to produce exopolysaccharides (Ivanova et al., Mikrobiologiya (1988), 57(4), 600-5); Kilbane, John J., II Gas, Oil, Coal, Environ. Biotechnol. 3, [Pap. IGT""s Int. Symp.], 3rd (1991), Meeting Date 1990, 207-26. Editor(s): Akin, Cavit; Smith, Jared. Publisher: IGT, Chicago, Ill.). Similarly, methanotrophs are known to accumulate both isoprenoid compounds and carotenoid pigments of various carbon lengths (Urakami et al., J. Gen. Appl. Microbiol. (1986), 32(4), 317-41). Although these compounds have been identified in methanotrophs, they have not been microbial platforms of choice for production because these organisms have very poorly developed genetic systems, thereby limiting metabolic engineering ability for chemicals.
A necessary prerequisite to metabolic engineering of methanotrophs is a full understanding, and optimization, of the carbon metabolism for maximum growth and/or product yield. In methanotrophic bacteria, methane is converted to biomolecules via a cyclic set of reactions known as the ribulose monophosphate pathway (RuMP) cycle. The RuMP pathway is comprised of three phases, each phase being a series of enzymatic steps. The first phase (fixation) is the aldol condensation of three molecules of C-1 (formaldehyde) with three molecules of pentose (ribulose-5-phospate) to form three molecules of a six-carbon sugar (fructose-6-phosphate) catalyzed by hexulose monophosphate synthase. This fixation phase is common to all methylotrophic bacteria using the RuMP pathway.
The second phase is termed xe2x80x9ccleavagexe2x80x9d and results in splitting of that 6-carbon sugar into two 3-carbon molecules. This may be achieved via two possible routes. Fructose-6-phosphate is either converted into fructose-1,6-biphosphate (FBP) by phosphofructokinase, and subsquently cleaved by FBP aldolase (FBPA) to 3-carbon molecules, or oxidized to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and ultimately cleaved to 3-carbon sugars by the enzyme catalyzed by KDPG aldolase. One of those 3-carbon molecules is recycled back through the RuMP pathway and the other 3-carbon fragment is utilized for cell growth.
In the third phase (the xe2x80x9crearrangementxe2x80x9d phase), the regeneration of 3 molecules of ribulose-5-phosphate is accomplished from the two remaining molecules of fructose-6-phosphate (from stage 1) and the one molecule of the 3-carbon sugar from stage 2. There are two possible routes to achieve the rearrangement. These routes in the rearrangement phase differ in that they involve either transaldolase (TA) or sedoheptulose-1,7-bisphosphatase (SB Pase).
In methanotrophs and methylotrophs, the RuMP pathway may occur as one of three variants. These are the KDPGA/TA, FBPA/SBPase and FBPA/TA pathways. However, only two of these variants are commonly found. These two pathways are the FBPA/TA (fructose bisphophotase aldolase/Transaldolase) or the KDPGA/TA (keto deoxy phosphgogluconate aldolase/transaldolase) pathway, wherein only the FBPA/TA pathway is exergonic (Dijkhuizen et al. (1992) The Physiology and biochemistry of aerobic methanol-utilizing gram negative and gram positive bacteria. In: Methane and Methanol utilizers. P. 149-Colin Murrell and Howard Dalton, Plenum Press NY). Available literature suggests that obligatory methanotrophic bacteria such as Methylomonas rely solely on the KDPGA/TA pathway (Entner-Douderoff Pathway), while facultative methylotrophs utilize either the FBPA/SBPase or the FBPA/TA pathway (Dijkhuizen et al. supra). Energetically, this pathway is not as efficient as the Embden-Meyerhof pathway and thus could result in lower cellular production yields, as compared to organisms that do use the latter pathway. Therefore, a more energy efficient carbon processing pathway would greatly enhance the commercial viability of the methanotrophic platform for the generation of materials.
The problem to be solved therefore is to discover genes encoding a more energetically efficient carbon flux pathway that would enable a methanotrophic bacterial strain to better able to serve as a platform for the production of proteins and carbon containing materials. Applicants have solved the stated problem by providing the genes encoding the carbon flux pathway in a strain of Methylomonas. This pathway contains not only the expected elements of the Entner-Douderoff Pathway (including the 2-keto-3-deoxy-6-phosphogluconate aldolase) but additionally contains the elements of the more energy efficient Embden-Meyerhof pathway, containing the fructose-1,6-biphosphate aldolase. This discovery will permit the engineering of methanotrophs and other organisms for the energy efficient conversion of single carbon substrates such as methane and methanol to commercially useful products in the food and feed and materials industries.
The present invention provides an isolated nucleic acid molecule encoding a Methylomonas sp carbon flux enzyme, selected from the group consisting of:
(a) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; and
(c) an isolated nucleic acid molecule that is complementary to (a) or (b).
Additionally the invention provides the gene products, encoded by the present invention and chimera made from the instant genes by operably linking the instant genes to suitable regulatory sequences. Similarly the invention provides transformed host cells expressing the instant genes or their chimera.
The invention additionally provides a method of obtaining a nucleic acid fragment encoding a carbon flux enzyme comprising:
(a) probing a genomic library with the nucleic acid fragment of the present invention;
(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of the present invention; and
(c) sequencing the genomic fragment that comprises the clone identified in step (b),
wherein the sequenced genomic fragment encodes a carbon flux enzyme.
Alternatively the invention provides a method of obtaining a nucleic acid fragment encoding a carbon flux enzyme comprising:
(a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19;
(b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);
wherein the amplified insert encodes a portion of an amino acid sequence encoding a carbon flux enzyme.
In another embodiment the invention provides a method of altering carbon flow through a methanotrophic bacteria comprising, over-expressing at least one carbon flux gene selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 in a methanotrophic strain such that the carbon flow is altered through the strain.
Additionally the invention provides a mutated gene encoding a carbon flux enzyme having an altered biological activity produced by a method comprising the steps of:
(i) digesting a mixture of nucleotide sequences with restriction endonucleases wherein said mixture comprises:
a) a native carbon flux gene;
b) a first population of nucleotide fragments which will hybridize to said native carbon flux gene;
c) a second population of nucleotide fragments which will not hybridize to said native carbon flux gene;
wherein a mixture of restriction fragments are produced;
(ii) denaturing said mixture of restriction fragments;
(iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase;
(iv) repeating steps (ii) and (iii) wherein a mutated carbon flux gene is produced encoding a protein having an altered biological activity.